The chemotaxis, or directed migration, of vascular smooth muscle cells (VSMCs) is critical in the development of atherosclerosis and post-angioplasty restenosis, yet the cytosolic signaling pathways regulating this event are still largely undetermined. Our laboratory has demonstrated that: 1) specifically-timed Cai elevations, which in turn activate Ca2+/ calmodulin-dependent protein kinase II (CaMK), are required for chemotaxis, and 2) that the responses of individual cells need be examined due to the asynchrony and rarity of migration (only 5-10% of cells undergo chemotaxis by 4 hours). The precise role of CaMK, however, has yet to be resolved. Therefore, the specific aim was to determine whether compartmentalization of CaMK, and its activity, occurs within cells, since discrete localization of a kinase (or its activity) could implicate its function and target. The intracellular distribution of CaMK was determined in both fixed and live individual VSMCs respectively using immunocytochemistry (with three antibodies specific for different epitopes on CaMK) and live-cell imaging (with AS2, a novel fluorescent probe for CaMK activity). Subcellular domains of CaMK exist within VSMCs with a certain pool of CaMK decorating the polymerized actin cytoskeleton. Dynamic shifts in the state of actin polymerization (which accompany chemotaxis) result in an apparent translocation between a "soluble" pool and an "immobilized" pool of CaMK. The CaMK that is localized to polymerized actin stress fibers is activated (above the basal state) in quiescent, non-migrating VSMCs. Reactive oxygen species may provide a mechanism for "local" maintenance of this cytoskeletal-associated CaMK activity during quiescence. In migrating VSMCs, activated CaMK is localized to the cortical shell of actin that develops during chemotaxis, which suggests that CaMK may play a critical role in modulating force generation associated with directed migration. Thus, discrete localization of CaMK may enable specific functional targeting and regulation of the cytoskeleton during both quiescence and chemotaxis.